High speed scanning system with acceleration tracking

ABSTRACT

Disclosed herein is a high throughput optical scanning device and methods of use. The optical scanning device and methods of use provided herein can allow high throughput scanning of a continuously moving object with a high resolution despite fluctuations in stage velocity. This can aid in high throughput scanning of a substrate, such as a biological chip comprising fluorophores. Also provided herein are improved optical relay systems and scanning optics.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.15/910,778, filed on Mar. 2, 2018, which claims the benefit of U.S.Provisional Application No. 62/467,048, filed on Mar. 3, 2017, theentire disclosures of which are hereby incorporated by reference intheir entireties.

FIELD OF THE INVENTION

The invention relates to methods and instruments for generating a stableimage of a continuously moving substrate in an optical system.

BACKGROUND OF THE INVENTION

Typical single-molecule, single-fluor sensitivity biological fluorescentoptical scanning systems require low noise cameras with long exposuretimes. These systems often require a high precision and stable imagingplatform situated on granite or equivalent. In addition, these systemsemploy “step and repeat” staging which necessitate high acceleration anddeceleration as well as high mass in order to achieve high throughput,stable imaging of multiple fields. To scan a large area chip (2000 mm2)in a short amount of time (−5 min) at high magnification requires frameimaging times shorter than step and repeat systems allow.

An “image on the fly” approach is needed to prevent a loss in throughputdue to stage accelerations and settling time inherent to the step andrepeat systems. Traditional image on the fly applications require samplestages that can provide near-constant (—+/−0.05%) velocity, and scanningoptics that image the sample as it moves. If the stage velocity is notnear-constant throughout its travel, then the scanning optics will notimage the exact same region of the sample as the stage moves. This canresult in a blurry image (e.g., with a pixel smear of—+/−3 pixels). Thisproblem is typically solved by utilizing expensive stages that providenear constant velocity by using heavy stages and powerful stage drives.Unfortunately, this adds to the cost of the product and makes itimpractical to use as a benchtop system.

Typical low-cost, compact and/or lightweight stages are built withcomponents that have various surface irregularities such as pits, burrs,machining grooves, divots and misshapen cavities. These irregularitiesusually result in velocity that is not constant. For instance, a burr ora divot in the ways of a stage will cause the stage to momentarily slowdown and then possibly speed up before returning to the velocity it hadbefore it encountered the irregularity. The velocity fluctuations of thestage make the use of these low-cost, smaller components incompatiblewith current image on the fly high throughput scanning approaches due tothe generation of unacceptable levels of image blur.

What is needed therefore, are improved scanning optics that increase thevelocity fluctuation tolerance, allowing an image with increasedstability (e.g., reduced pixel smear) to be obtained using image on thefly scanning with single fluor sensitivity in smaller, lightweight, andlow-cost optical scanning systems.

SUMMARY OF THE INVENTION

The instant invention is based, at least in part, on the discovery ofnew methods and devices to reduce pixel smear in the imaging of anobject on a moving stage.

Accordingly, provided herein is an optical scanning system for imaging amoving substrate, comprising a stage, said stage capable of moving alongan axis, said stage configured to hold a substrate comprising aplurality of fields; an objective lens; a camera capable of acquiring animage of one of said plurality of fields through the objective lens,said image acquired via an optical path defined from one of saidplurality of fields through said objective lens to said camera duringacquisition of said image; a velocity tracking mirror mounted along saidoptical path; a first electrical motor operably coupled to said velocitytracking mirror to adjust the angle of the tracking mirror along saidaxis of stage movement in said optical path; a controller moduleoperably coupled to said first electrical motor to send a first drivingsignal to said first electrical motor, wherein said first driving signalis a function of a velocity measurement of the stage movement along saidaxis; an acceleration tracking mirror mounted along said optical path; asecond electrical motor operably coupled to said acceleration trackingmirror to adjust the angle of the acceleration tracking mirror alongsaid axis of stage movement in said optical path, wherein saidcontroller module is operably coupled to said second electrical motor tosend a second driving signal to said second electrical motor, whereinsaid second driving signal is a function of the change of the stagevelocity along said axis.

In some embodiments, the first or second driving signal is an electricalsignal. In some embodiments, the first driving signal comprises anon-sinusoidal waveform. In some embodiments, the non-sinusoidalwaveform is a sawtooth wave. In some embodiments, the first electricalmotor is a galvanometer. In some embodiments, the second electricalmotor is a piezoelectric actuator. In some embodiments, the firstelectrical motor or said second electrical motor are dual axes motors.

In some embodiments, the system further comprises a linear displacementsensor operably coupled to said controller module to send a signalcomprising a positional measurement of said substrate or said stage tosaid controller module. In some embodiments, the linear displacementsensor is a linear encoder.

In some embodiments, the first driving signal is a function of avelocity determined from said positional measurement. In someembodiments, the second driving signal is a function of a change invelocity determined from said positional measurement.

In some embodiments, the first or second signal comprises a waveformthat is a function of the field scan frequency. In some embodiments, thefirst or second signal comprises a waveform that is a function of theimaging duty cycle.

In some embodiments, the movement of said velocity tracking mirror andsaid acceleration tracking mirror reduce a tracking error of said fieldby said camera as compared to without movement of said accelerationtracking mirror. In some embodiments, the tracking error is reduced toless than 0.1%. In some embodiments, the tracking error is reduced toless than 1 pixel.

In some embodiments, the velocity tracking mirror and the accelerationtracking mirror are adjacent components along said light path.

In some embodiments, the system comprises a plurality of cameras. Insome embodiments, the system further comprises a beam splitter mountedalong said light path, wherein said beam splitter is mounted along saidlight path after said velocity tracking mirror and acceleration trackingmirror and before said plurality of cameras.

In some embodiments, the system further comprises an illumination path,said illumination path extending from an illumination element to one ofsaid plurality of fields.

In some embodiments, the illumination element comprises an excitationlaser operably mounted to transmit an excitation light to said field,wherein said optical path comprises fluorescent light emitted from saidfield to said camera. In some embodiments, the excitation light is nottransmitted to said camera. In some embodiments, the illuminationelement comprises an illumination light operably mounted to transmit anillumination light to said field. In some embodiments, the illuminationlight is mounted underneath the field such that the optical pathcomprises light transmitted through said field and to said camera. Insome embodiments, the illumination light is mounted above or transverseto said field such that the optical path comprises light reflected bysaid field and to said camera.

In some embodiments, the system further comprises a third electricalmotor operably mounted to the objective lens to move the objective lensalong said optical path, thereby maintaining said field in focus. Insome embodiments, the third electrical motor is operably connected tosaid controller module to receive a third driving signal that is afunction of the movement of the field out of the focal plane, such thatthe objective lens is moved to maintain said field in focus by saidcamera.

In some embodiments, the system further comprises at least oneadditional pair of mirrors comprising a second velocity tracking mirrorand a second acceleration tracking mirror, wherein said mirrors areoperably mounted to said device to reduce a tracking error of said fieldby said camera along a different axis.

Also provided herein is a method of imaging a plurality of fields on amoving substrate, comprising providing an optical scanning systemcomprising a moveable stage holding a substrate comprising a pluralityof fields, a camera, an objective lens, a velocity tracking mirror, andan acceleration tracking mirror; moving said moveable stage along anaxis, thereby moving said substrate comprising a plurality of fieldsalong said axis; and concurrent with said movement, capturing an imageof one of said plurality of fields passing through said objective lensusing said camera, wherein said image of said field is stabilized duringsaid image capture by rotating said velocity tracking mirror as afunction of a velocity of the moveable stage along said axis, androtating said acceleration tracking mirror as a function of a change inthe velocity of the moveable stage along said axis.

In some embodiments, the method of imaging a plurality of fields on amoving substrate further comprises obtaining a measurement of thevelocity of said moveable stage, said substrate, or said field alongsaid axis, and adjusting said first driving signal as a function of saidvelocity. In some embodiments, the method of imaging a plurality offields on a moving substrate further comprises determining a change invelocity of said moveable stage from a plurality of velocitymeasurements, and adjusting said second driving signal as a function ofsaid change in velocity.

In some embodiments, the rotation of the velocity tracking mirror or theacceleration tracking mirror is performed based on said measuredvelocity or said measured change in velocity. In some embodiments, thefirst driving signal is a function of an anticipated velocity of saidstage. In some embodiments, the second driving signal is a function ofan anticipated change in velocity of said stage.

In some embodiments, the velocity tracking mirror is operably coupled toa first electric motor. In some embodiments, the first electric motor isa galvanometer. In some embodiments, the optical scanning systemcomprises a controller module, and wherein said first electric motor isoperably coupled to said controller module.

In some embodiments, rotating said velocity tracking mirror comprisessending a first driving signal from said controller module to said firstelectric motor. In some embodiments, the first driving signal is afunction of a measured or predetermined velocity of the substrate.

In some embodiments, the acceleration tracking mirror is operablycoupled to a second electric motor. In some embodiments, the electricmotor is a piezoelectric actuator. In some embodiments, the electricmotor is operably coupled to said controller module. In someembodiments, the acceleration tracking mirror comprises sending a seconddriving signal from said controller module to said second electricmotor.

In some embodiments, the second driving signal is a function of ameasured or predetermined change in the velocity of the substrate. Insome embodiments, the second driving signal is a function of a deviationof said velocity from the velocity used to determine said first drivingsignal.

In some embodiments, the velocity tracking mirror and the accelerationtracking mirror are adjacent.

In some embodiments, the movement of said velocity tracking mirror andsaid acceleration tracking mirror reduce a tracking error of said fieldby said camera as compared to without movement of said accelerationtracking mirror. In some embodiments, the tracking error is reduced toless than 0.1%. In some embodiments, the tracking error is reduced toless than 1 pixel.

In some embodiments, the method further comprises adjusting the locationof the objective lens along the optical path to maintain said field infocus during said image capture. In some embodiments, the adjustment ofthe objective lens maintains an intensity jump between two adjacentpixels of said image of greater than 50%, 60%, 70%, 80%, or 90%.

In some embodiments, the optical scanning system further comprises asecond velocity tracking mirror and a second acceleration trackingmirror, further comprising, concurrent with said movement of saidmoveable stage and said image capture of one of said plurality offields: rotating said second velocity tracking mirror as a function of avelocity of the moveable stage along a second axis, and rotating saidsecond acceleration tracking mirror as a function of a change in thevelocity of the moveable stage along said second axis therebystabilizing imaging of said field for at least two axes simultaneously.

In some embodiments, the method further comprises rotating each of aplurality of pairs of velocity tracking and acceleration trackingmirrors to stabilize an image for a corresponding plurality of distinctaxes.

In some embodiments, the frequency of image capture is at least 20 Hz,40 Hz, 60 Hz, 80 Hz, 100 Hz, 120 Hz, 140 Hz, 160 Hz, 180 Hz, or 200 Hz.In some embodiments, the duty cycle of the image capture is at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. In some embodiments, the dutycycle of the image capture is at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80% or 90%.

Also provided herein is a method of reducing positioning error in animage obtained from a moving stage, comprising: measuring a velocity ofsaid moving stage; determining an error correction term from saidmeasured velocity as a function of the difference between the measuredvelocity of said stage and an anticipated velocity of said stage;generating a driving signal as a function of said error correction term;and sending said driving signal to an electrical motor, wherein saidelectrical motor is operably connected to a tracking mirror to actuaterotation of said tracking mirror. In some embodiments, the motor is agalvanometer or a piezoelectric actuator.

Also provided herein is an optical scanning system for imaging a movingsubstrate, comprising: a stage, said stage capable of moving along anaxis, said stage configured to hold a substrate comprising a pluralityof fields; an objective lens; a camera capable of acquiring an image ofone of said plurality of fields through the objective lens, said imageacquired via an optical path defined from one of said plurality offields through said objective lens to said camera during acquisition ofsaid image; a motion tracking mirror mounted along said light path; anelectric motor operably coupled to said motion tracking mirror toactuate angular motion of the tracking mirror along said axis of stagemovement in said optical path; and a controller module operably coupledto said electric motor to send a driving signal to said electric motor,wherein said controller module is capable of generating said drivingsignal as a function of a velocity fluctuation of said stage orsubstrate movement along said axis.

In some embodiments, the device comprises a velocity sensor inelectrical communication with the controller module, said velocitysensor capable of detecting positional or velocity information of thesubstrate or stage and sending said information to the controllermodule, wherein said controller module is configured to generate saiddriving signal as a function of the velocity signal received from thevelocity sensor.

In some embodiments, the sensor is a linear encoder. In someembodiments, the linear encoder is a non-interferometric encoder. Insome embodiments, the linear encoder is optical, magnetic, capacitive,inductive, or uses an Eddy current. In some embodiments, the sensor iscalibrated for velocity feedback across the moveable stage.

In some embodiments, the driving signal is a function of both apre-determined velocity and a measured velocity. In some embodiments,the stage comprises a mechanical bearing positioned to facilitatemovement of said stage along an axis. In some embodiments, the objectivelens has a magnification selected from the group consisting of: 5×, 10×,20×, 30×, 40×, 50×, 60×, 70×, 80×, 90×, or 100×.

Also provided herein is a method of imaging a plurality of fields on amoving substrate, comprising: providing an optical scanning systemcomprising a moveable stage holding a substrate comprising a pluralityof fields, an objective lens, a camera, a motion tracking mirror, and anelectric motor operatively coupled to said motion tracking mirror toeffect movement of said motion tracking mirror to track said movement ofsaid moveable stage along said axis during an image capture, and toreturn said motion tracking mirror to an initial position after saidimage capture; moving said moveable stage along an axis, thereby movingsaid substrate comprising a plurality of fields along said axis; andgenerating an image for each of M fields of said substrate, performingat least M image capture cycles during movement of said moveable stagealong said axis, each cycle comprising: providing a cycle M drivingsignal to an electric motor to control movement of said tracking mirrorto track the velocity of said moveable stage along said axis; capturingan image of said field while said tracking mirror is tracking saidmoving stage; and determining an average velocity of said field, whereinsaid average velocity is used to generate a cycle M+1 driving signal tocontrol movement of said electric motor during cycle M+1.

In some embodiments, the frequency of image capture is at least 20 Hz,40 Hz, 60 Hz, 80 Hz, 100 Hz, 120 Hz, 140 Hz, 160 Hz, 180 Hz, or 200 Hz.In some embodiments, the duty cycle of the image capture is at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. In some embodiments, the dutycycle of image capture is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80% or 90%.

In some embodiments, the method further comprises performing an initialcycle to determine an average velocity of said field, wherein no imagecapture occurs. In some embodiments, the M+1 driving signal comprises acorrection term that is a function of the difference between a measuredvelocity and a desired velocity of said moveable stage along said axis.In some embodiments, determining said average velocity of said fieldcomprises measuring one or more positions of a field at a time. In someembodiments, the average velocity of said field further comprisescomparing said measured position of said field at said time with apreviously measured position and time of another field.

In some embodiments, the velocity feedback loop duration from positionalmeasurement of field M to providing said M+1 driving signal is no morethan 100 ms and could be as low as 2 ms, 90 ms, 80 ms, 70 ms, 60 ms, 50ms, 40 ms, or 30 ms. In some embodiments, the average velocity isdetermined by collecting information about the position of the substrateat a frequency of no more than 250 kHz, 200 kHz, 150 kHz, 100 kHz, 50kHz, 20 kHz, 10 kHz, 5 kHz, 2 kHz, 1000 Hz, 500 Hz, 240 Hz, 120 Hz, 60Hz, or 30 Hz.

In some embodiments, the generated image has a pixel smear of no morethan +/−one pixel. In some embodiments, the pixel comprises across-sectional distance along said axis of about 150 nm on saidsubstrate.

In some embodiments, the image is generated from a substrate moving at avelocity in a range from 100 p.m./second to 1,000 mm/second. In someembodiments, the movement of said moveable stage along said axiscomprises velocity fluctuations in the range of 0.1% to 1% of theaverage velocity.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages will beapparent from the following description of particular embodiments of theinvention, as illustrated in the accompanying drawings in which likereference characters refer to the same parts throughout the differentviews. The drawings are not necessarily to scale, emphasis insteadplaced upon illustrating the principles of various embodiments of theinvention.

FIG. 1 is a diagram of components of an optical scanning device along anoptical path from a substrate to a detector comprising an accelerationtracking mirror and a velocity tracking mirror (i.e., a dual mirrorembodiment) according to an embodiment of the invention.

FIG. 2 is a representation of a sawtooth waveform to control the motionof a moving stage tracking mirror, such as a velocity tracking mirror,and its correlation to the change in position of a moveable stage alongan axis over time according to an embodiment.

FIG. 3 provides an example of waveforms that can be used to generatedriving signals for a velocity tracking mirror and an accelerationtracking mirror (in a dual mirror embodiment) to stabilize an image of amoving substrate based on a measured or anticipated stage velocity errorand an anticipated stage velocity.

FIG. 4 is a diagram of components of an optical scanning device along anoptical path from a substrate to a detector comprising a single motiontracking mirror (i.e., a single-mirror embodiment) according to anembodiment of the invention.

FIG. 5 provides an example of waveforms that can be used to generatedriving signals for a motion tracking mirror (in a single mirrorembodiment) to stabilize an image of a moving substrate based on ameasured or anticipated stage velocity error and an anticipated stagevelocity.

FIG. 6 provides a schematic of one possible implementation of a fieldlevel feed-forward mechanism to provide a correction term to adjust adrive signal provided to a mirror of the device capable of moving inresponse to velocity fluctuations of the substrate or moveable stage.

FIGS. 7A and 7B are diagrams of components of a controller module andits connections to certain components of the device, including theadjustable tracking mirror(s) and substrate or moveable stage positionsensing devices. Connections are indicated by arrows. A solid arrowindicates a signal sent from the controller module to the respectivecomponent. Dotted arrow indicates the path for measurement of velocityfluctuations of the stage or substrate and translation into a drivingsignal that controls the motor operably connected to an acceleration ormotion tracking mirror. Dashed arrows indicate the movement of lightfrom along an optical path among components of the optical scanningsystem. FIG. 7A is related to a dual mirror embodiment, while FIG. 7B isrelated to a single mirror embodiment.

FIG. 8A provides a flowchart of a method of operating a dual trackingmirror embodiment of a device to capture a stabilized image of a fieldof a moving substrate.

FIG. 8B provides a flowchart of a method of operating a single trackingmirror embodiment of a device to capture a stabilized image of a fieldof a moving substrate.

FIGS. 9A and 9B illustrate an example of a pixel smear of an image of +1and +2 respectively. Each square represents a pixel.

FIG. 10 illustrates an embodiment of the optical scanning system,including components for controlling and storing data from the system.

DETAILED DESCRIPTION

The details of various embodiments of the invention are set forth in thedescription below. Other features, objects, and advantages of theinvention will be apparent from the description and the drawings, andfrom the claims.

As used herein, the term “objective lens” refers to an element or groupof elements, in an optical scanning system, that comprises one or morelenses and is configured and operative to magnify an electromagnetic(e.g., such as optical) signal. In some embodiments, an objective lenshas a large numerical aperture (NA), such as an NA in a range between0.6 and 1.5 and performs imaging via air immersion or liquid immersion(e.g., such as water, oil, or other immersion fluids). In variousembodiments, an objective lens may have a focal length in the range from2 mm to 25 mm.

As used herein, the term “substrate” refers to an object having amultitude of distinct features that are targets for imaging. Thesefeatures may or may not be arranged in a spatially uniform pattern. Forexample, in some embodiments a substrate comprises a non-planarstructure with a surface, such as a bead or a well, to which targetbiomolecules have been attached as the target features. In anotherexample, in some embodiments a substrate comprises an array chip. Anarray chip (i.e., an array, microarray, or chip) refers to a solid phasesupport having a surface, preferably but not exclusively a planar orsubstantially planar surface, that carries attachment sites to whichtarget biomolecules (e.g., such as proteins or nucleic acids) have beenattached as the target features.

As used herein, the term “field” refers to an area of a substratecapable of being captured within a single image by a camera. A field onthe substrate is related to the field of view of the camera. An entiresubstrate may be scanned by taking images of a plurality of fields on asubstrate.

As used herein, the term “optical path” or “light path” refers to thepath of light or other electromagnetic radiation from a source to thecamera sensor. Manipulation of the optical path by mirrors along theoptical path enable the capture of a still image from a continuouslymoving substrate with random or systematic velocity fluctuations.

As used herein, the term “scanning” refers to operations to observe andrecord the status of a substrate.

As used herein, the term “velocity tracking mirror” refers to a mirrorconfigured to track the movement of a substrate at a velocity. Thisvelocity may be fixed or variable. The velocity may be predetermined, ormay include systematic or random error in the velocity.

As used herein, the term “velocity tracking error” refers to an error inthe tracking of a substrate or stage velocity by the velocity trackingmirror. In some embodiments, this is the result of a deviation in thevelocity of the substrate from the velocity being tracked by thevelocity tracking mirror.

As used herein, the term “acceleration tracking mirror” refers to amirror that is operably connected to an optical scanning system torotate in response to a nonlinearity, such as a systematic or randomerror in stage velocity, or any other deviations from an expected orconstant stage velocity. In some embodiments, the acceleration trackingmirror is paired with a velocity tracking mirror to provide a stillimage of a moving substrate with reduced pixel smear.

As used herein, the term “electrical motor” refers to a device thatconverts an electrical signal to a physical movement, such as a motorthat rotates in response to electrical energy. In some embodiments, theelectrical motor provides a rotation mechanism for rotating a velocitytracking mirror or an acceleration tracking mirror. The electrical motorcan be operably linked to a controller module that sends an electricalsignal or driving signal to effect controlled movement of the electricalmotor. An electrical motor may be a galvanometer or a piezoelectricactuator. As used herein, a “galvanometer” refers to a coil in amagnetic field that moves in response to an electrical signal. This canact as an electrical motor to actuate rotary motion of a trackingmirror. As used herein, the term “piezoelectric actuator” refers to atype of electric motor based upon the change in shape of a piezoelectricmaterial when an electric field is applied. Although electrical motorsare referred to in this specification as a preferred embodiment, otherdevices to provide actuation of components of the parts of the inventiondescribed herein, such as those based on hydraulics, pneumatics, ormagnetic principles may also be used.

As used herein, the term “controller module” refers to one or morecomponents in the device that provide control over components of theoptical scanning system. In particular, the controller module includesdevices that control movement of the electrical motors operablyconnected to one or more tracking mirrors. Thus, the controller modulegenerates and transmits a driving signal to these electrical motors. Thedriving signal may be generated from a pre-programmed or observed stageor substrate motion. The driving signal may be generated frominformation collected by a position or velocity sensor, such as anencoder, and used to generate a velocity measurement that is thentranslated into a responsive driving signal to control movement of oneor more tracking mirrors.

As used herein, the term “electrical signal” or “driving signal” refersto a controlled amount of energy sent to an electrical motor that themotor transforms into physical movement. For example, a galvanometer canaffect rotation of a mirror to track a moveable stage and to return toits original position after imaging is complete by sending a drivesignal that resembles a sawtooth wave.

As used herein, the term “duty cycle” refers to the percent of time atracking mirror is tracking the stage and the camera is imaging thefield (as opposed to flyback time, where the tracking mirror isreturning to its initial position).

As used herein, the term “imaging frequency” or “image capturefrequency” refers to the frequency of image capture of fields on asubstrate.

As used herein, the term “pixel smear” refers to a measure of the spreadof a pixel along an axis due to movement of an imaged object duringimage capture. A high amount of pixel smear will generate an image thatis less sharp and has a higher amount of blur. In some embodiments,pixel smear is generated due to velocity fluctuations that are notcompensated for in the optical path or in the movement of one or moretracking mirrors. Provided herein, in some embodiments, are devices andmethods for capturing an image of a continuously moving substrate on amoveable stage with velocity fluctuations wherein the amount of pixelsmear along the primary axis of movement of the substrate is mitigatedby the rotation of one or more tracking mirrors along the optical path.

As used herein, the term “logic” refers to a set of instructions which,when executed by one or more processors (e.g., CPUs) of one or morecomputing devices, are operative to perform one or more functionalitiesand/or to return data in the form of one or more results or of inputdata that is used by other logic elements and/or by elements thatcontrol the operation of mechanical devices (e.g., such as servos andthe like). In various embodiments and implementations, any given logicmay be implemented as one or more software components that areexecutable by one or more processors (e.g., CPUs), as one or morehardware components such as Application-Specific Integrated Circuits(ASICs) and/or Field-Programmable Gate Arrays (FPGAs), or as anycombination of one or more software components and one or more hardwarecomponents. The software component(s) of any particular logic may beimplemented, without limitation, as a standalone software application,as a client in a client-server system, as a server in a client-serversystem, as one or more software modules, as one or more libraries offunctions, and as one or more static and/or dynamically-linkedlibraries. During execution, the instructions of any particular logicmay be embodied as one or more computer processes, threads, fibers, andany other suitable run-time entities that can be instantiated in thehardware of one or more computing devices and can be allocated computingresources that may include, without limitation, memory, CPU time,storage space, and network bandwidth.

Optical Scanning System and Methods of Use

Provided herein is a lightweight, cost-effective system for high framerate image capture of portions or fields of a substrate with a highsensitivity while the substrate is moving on a moveable stage. Thisoptical scanning system is capable of high speed, single molecule,single-fluor imaging, which to date has only been provided by heavy andexpensive systems requiring precise control of stage movement, orthrough slower step and repeat optical scanning systems. The opticalscanning system provided herein can be used as an image on the flysystem (continuously moving stage) by using scanning optics whichcompensate for stage velocities that vary by 1% to 10% (typicallyresulting in image blur of at least several pixels). This compensationcan result in an image equivalent of a tracked staged velocity withfluctuations less than 0.1% or a pixel smear of an image of no more than+/−1 pixel. Therefore, the scanning optics disclosed herein provide asystem to compensate for velocity error (i.e., velocity fluctuations),such as localized accelerations and decelerations of a moveable stage orsubstrate, to provide a stabilized image field to a camera duringimaging of a continuously moving moveable stage to reduce pixel smear.

The optical scanning system disclosed herein uses rotatable scanningoptics and a control system to stabilize an optical path between asubstrate and a detector while a substrate is in motion. The rotate ablescanning optics rotate in response to stage velocity and stage velocityfluctuations (or to substrate velocity and substrate velocityfluctuations). Scanning optics provided by one embodiment of an opticalscanning system with dual tracking mirrors are shown in FIG. 1. In thisembodiment, the optical scanning system comprises a moveable stage 110configured to move a mounted substrate 120 along an axis. The substrate120 comprises one or more fields 121 that are individually imaged by theoptical scanning system as the stage is continuously moving. Thesubstrate is illuminated by an illumination mechanism (not shown), andlight from the substrate travels along an optical path through theobjective lens 130. The image of the moving substrate is stabilized withrespect to an image sensor by a velocity tracking mirror 140 and anacceleration tracking mirror 150. An image of the field 121 is capturedby a camera 160 comprising an image sensor. The velocity tracking mirror140 is configured to rotate about an axis parallel to the plane of theimage field. The rotation of the velocity tracking mirror 140 adjuststhe optical path to stabilize the image of a field moving at apredetermined velocity during an image capture by the camera 160. Theacceleration tracking mirror 150 is configured to rotate about an axisparallel to the plane of the image field. The acceleration trackingmirror 150 rotates as a function of velocity fluctuations (i.e.,accelerations) of the moving stage or substrate. This rotation adjuststhe optical path to stabilize an image by compensating for velocityfluctuations in the movement of the stage and/or substrate along anaxis.

The optical scanning system in several embodiments is configured toimage a continuously moving object, such as a substrate mounted on amoveable stage, in a scanning fashion. In such embodiments, a substrateis typically mounted (or otherwise placed) on a moveable stage that iscoupled to one or more mechanisms (e.g., motors or other actuators) thatcan continuously move the substrate under an objective lens while acamera captures an image of a field of the substrate. The moveable stageis configured and operative to move the substrate along a direction thatis normal to the optical axis of the objective lens. In someembodiments, the axis of movement of the moveable stage is orthogonal tothe operation of autofocus-types of mechanisms, which generally move animaged object and/or an objective along the optical axis of theobjective lens.

In various embodiments, the velocity of the moveable stage may be in arange from 0.1 mm per second to 1000 mm per second (or greater). In someembodiments, the velocity of the moveable stage may be in a range from10 mm per second to 100 mm per second. In some embodiments the moveablestage (and therefore the substrate mounted thereon) can be configured tomove at a constant velocity, although the stage is still subject tovelocity fluctuation errors that are compensated for by the opticalsystems provided herein. In some embodiments, the moveable stage movesat a velocity of 10 to 50 mm per second. In some embodiments, thevelocity of the moveable stage is about 25 mm per second. In otherembodiments, the moveable stage can be configured to move withnon-constant velocity. This non-constant velocity can also be subject tofluctuation errors that are compensated for by the optical systemsprovided herein.

In some embodiments, mechanisms may be used to facilitate the motion ofthe moveable stage at a given desired velocity. Such mechanisms maycomprise one or more components that cause motion (e.g., such as linearmotors, lead screws, screw motors, speed screws, etc.) and one or morecomponents (e.g., such as various types of bearings) that reducefriction.

For example, in some embodiments, a moveable stage may use metalbearings (e.g., such as ball bearings, cylinder bearings, cross-rollerball bearings, etc.) that have repeatability of several microns tofacilitate motion of the moveable stage at a given desired velocity.Repeatability is fundamentally the effect of rolling a metal bearing inoil—as the metal bearing rolls it bounces, and such bouncing introducesjitter in the motion of the object that is being moved on the bearings.The “repeatability” of such motion can be uniform only above a certainrange because any two metal bearings can bounce in the same way onlywithin a certain tolerance. Thus, embodiments that use ball bearingstypically have greater velocity fluctuations, and thus introduce imageblur (e.g., pixel smear). However, stages using ball bearings provideseveral advantages, including that they are lighter, smaller, andcheaper than comparable air bearing stages. Thus, provided hereinaccording to some embodiments are improved scanning optics to reduceimage blur or pixel smear due to moveable stage velocity fluctuations,including stages with ball bearings or other components that providemotion subject to some velocity fluctuations.

In some embodiments, the velocity of the moveable stage fluctuates fromthe intended velocity by more than 0.1% during continuous opticalscanning. In some embodiments, the velocity of the moveable stagefluctuates from the intended velocity by more than 0.5% duringcontinuous optical scanning. In some embodiments, the velocity of themoveable stage fluctuates by between 0.1% and 1% during continuousoptical scanning. In some embodiments, the optical scanning systemprovided herein reduces an image blur or pixel smear from a moveablestage with a velocity fluctuation of between 0.1% and 1% to less than0.1%. In some embodiments, the pixel smear for a stabilized image isless than +/−1 pixel. In some embodiments, the moveable stage isconfigured to move a substrate in a continuous motion in a first knownlateral direction with respect to the objective lens while a camera witha two dimensional full-frame electronic sensor produces thetwo-dimensional image. In some embodiments, the moveable stage isconfigured to move in a continuous serpentine fashion to image aplurality of rows or columns of fields on a substrate.

In some embodiments, a substrate is mounted (or otherwise placed) on amoveable stage. In some embodiments, the substrate comprises an arrayhaving target biomolecules disposed thereon. In some embodiments, thesubstrate comprises a multitude of distinct features that are targetsfor imaging. e.g., such as array chips. In some embodiments, thesubstrate comprises a randomly positioned array of targets for imaging.

In some embodiments, the substrate comprises a multitude of distinctfeatures that are targets for imaging. For example, in some embodimentsa substrate comprises a non-planar structure with a surface, such as abead or a well, to which target biomolecules have been attached as thetarget features. In some embodiments, a substrate comprises an arraychip. In some embodiments, the array chip is a solid phase supporthaving a surface, e.g., a planar or substantially planar surface, thatcarries attachment sites to which biomolecules are attached as thetarget features. In some embodiments, the attachment sites on the arraychip may be arranged in an ordered pattern or in random fashion. In someembodiments, the attachment sites are configured to have dimensionssuitable for the attachment of target biomolecules. An attachment siteis thus spatially defined and is not overlapping with other sites; thatis, the attachment sites are spatially discrete on the array chip. Whenattached to the attachment sites, the biomolecules may be covalently ornon-covalently bound to the array chip.

In some embodiments, the substrate is a biochip. In some embodiments,the biochip comprises high throughput microfluidics. In someembodiments, the biochip comprises biomolecules for detection of singlemolecules from a sample. In some embodiments, the substrate comprises anarray having target nucleic acids disposed thereon. In anotherembodiment, the substrate comprises a multitude of distinct featuresthat are targets for imaging.

In some embodiments the attachment sites on a substrate are divided intofields that are each imaged separately. A typical substrate may bedivided into hundreds or thousands of fields that are arranged in arectangular pattern of rows and columns. (For example, the rows andcolumns of fields may include track regions that are alignedsubstantially along a horizontal dimension and a vertical dimension,respectively).

In such embodiments, the techniques described herein provide forscanning and imaging a substrate field by field. In one example, anoptical scanning system images a substrate in a scanning fashion (asdescribed herein) while the moveable stage is moving the substrate alonga y-direction in a plane and/or axis that is substantially normal to theoptical axis of the objective lens. In this example, the opticalscanning system ceases imaging when the end of the column of field(s)being imaged is reached in order to allow the moveable stage to positionthe substrate for imaging of the next column of field(s). In anotherexample, an optical scanning system images a substrate in a scanningfashion (as described herein) while the moveable stage is moving thesubstrate backward and forward in a serpentine fashion (e.g., along ay-direction) in a plane that is substantially normal to the optical axisof the objective lens. In this example, the optical scanning systemimages a column of field(s) while the moveable stage is moving thesubstrate in one direction and then images the next/adjacent column offield(s) while the moveable stage is moving/returning the substrate inthe opposite direction, e.g., the optical scanning system images thesubstrate by effectively traversing the columns of fields in acontinuous serpentine fashion.

The objective lens of the optical scanning system is configured andoperative to image a substrate or a portion thereof onto the camera. Insome embodiments, the objective lens is an element or group of elements,in an optical scanning system, that comprises one or more lenses and isconfigured and operative to magnify an electromagnetic (e.g., such asoptical) signal. In some embodiments, an objective lens has a largenumerical aperture (NA) (e.g., NA in a range between 0.6 and 1.5) andperforms imaging via air immersion or liquid immersion (e.g., such aswater, oil, or other immersion fluids). In various embodiments, anobjective lens may have a focal length in the range from 2 mm to 40 mm.The objective lens can be an off-the-shelf microscope objective or acustom-designed, multi-element optical component. In some embodiments,the objective lens is configured to image at least a two-dimensionalportion of a substrate onto the two dimensional full-frame electronicsensor of the camera to produce a two-dimensional image.

The magnification of an objective lens is the ratio of the size of animage space pixel (i.e., a camera pixel) to the actual size of theobject space area that corresponds to the image space pixel as observedby the camera. For example, a magnification of 16× allows a camera using81.tm pixels to observe 500 nm object space pixels. In some embodiments,the objective lens has a magnification from 4× to 100×. In someembodiments, the objective lens has magnification of 20× to 50×. In someembodiments, the objective lens has a magnification of 40×.

In some embodiments, the objective lens is operably connected to anelectrical motor for positioning the objective lens to allowauto-focusing. In some embodiments, the device comprises a focusingsensor. In some embodiments, the device comprises an array of focusingsensors.

In some embodiments, auto-focus mechanisms used are based on opticalsensing methods. In some embodiments, auto-focusing is performed byimage content analysis. In some embodiments, autofocusing is performedby obtaining multiple images of the substrate at multiple focaldistances, determining an optimal focal distance for each of the images,and using a feedback loop to adjust the focal distance.

Autofocusing can be performed by directing a laser beam at thesubstrate, measuring a reflection of the laser beam off the substrate toprovide a reference point, and using a feedback loop to adjust the focaldistance. In some embodiments, non-optical types of non-contact positionsensors are used. These sensors are capable of making position readingswith high bandwidth and a tracking precision of 0.1 p.m. or less. Insome embodiments, capacitive position sensors may be used (see, e.g., US2002/0001403, whose disclosure is incorporated herein by reference).

In some embodiments, autofocus of the objective lens is achieved in lessthan 100 ms. In some embodiments, the range of autofocus provided by thedevice is +/−200 p.m.

In some embodiments, the optical scanning device comprises an activeautofocus system that measures distance to the subject independently ofthe optical system, and subsequently adjusts the objective lens tocorrect focus. In some embodiments, a passive autofocus system thatdetermines correct focus by performing passive analysis of the imagethat is entering the optical system is used. Passive autofocusing can beachieved, for example, by phase detection or contrast measurement.

In some embodiments, the optical scanning system comprises a cameracapable of capturing a 2-dimensional still image of a field of thesubstrate while the substrate is being moved by the moveable stage. Insome embodiments, the optical scanning system comprises a full-framecamera. In some embodiments, the full-frame camera is a ComplementaryMetal-Oxide Semiconductor (CMOS) camera. These full frame cameras havehigh speed, high resolution, and low cost. Furthermore, they arecompatible with the optical scanning system for capturing an image of acontinuously moving substrate at a high resolution. In some embodiments,the camera is a scientific CMOS (sCMOS) camera. In some embodiments, thecamera is a non-CMOS camera capable of operating in full-frame mode.

The optical scanning systems described herein are configured to use fastcameras in conjunction with a scanning optics (e.g., single mirror ordual mirror embodiments) in order to achieve continuous exposure of astill image while the substrate being imaged is moving. In someembodiments, the size (length and/or width) of a camera pixel is in arange from 5 μm to 10 p.m., preferably but not exclusively in the rangeof 6-8 p.m. In some embodiments, the size of a camera pixel is 6.5 p.m.In some embodiments, the camera comprises an imaging sensor on the rangeof 15×15 mm to 10×10 mm.

In various embodiments, the optical scanning systems described hereinare configured to scan a continuously moving substrate (e.g., such as anarray chip) by using fast cameras that do not move the image through thecamera, e.g., such as non-TDI cameras and other cameras (including TDIcameras) that operate in full-frame 2D mode. CMOS cameras are an exampleclass of such cameras. CMOS cameras typically use an active-pixel sensor(APS) that is an image sensor comprising of an integrated circuitcontaining an array of pixels, where each pixel includes a photodetectorand an active amplifier.

A high-speed camera may be defined in terms of the number of pixels thatthe camera can expose in a unit of time. For example, the speed of thecamera may be defined by the mathematical product of the number ofpixels in the field of view and the frames per second that the cameracan take. Thus, a camera with a field of view of 5.5 megapixels (e.g., aview of 2560 pixels by 2160 pixels) running at 100 frames per second(fps) would be able to expose 550 megapixels per second; thus, suchcamera is termed herein as a “550” megapixel camera. Examples of suchcameras include, without limitation, CMOS, sCMOS, and similar cameras.In various embodiments, the optical scanning systems described hereinmay use cameras in the range from 10 megapixels to 2500 megapixels. Insome embodiments, the camera comprises a 2-dimensional, full frameelectronic sensor.

Scanning optics described herein as part of the optical scanning systemcan include single tracking mirror and dual tracking mirror embodimentshaving one or more rotatable mirrors affixed along an optical path ofthe system between the imaged object and the camera. In a dual trackingmirror embodiment, two sets of scanning optics are used, each able tomove in concert to track the motion of a moveable stage along an axisduring imaging. A first scanning optic (e.g., a velocity trackingmirror) is used to track the movement of a stage at an anticipatedvelocity or velocity pattern to enable imaging of a field by a camerawhile the field is in motion. A second scanning optic (e.g., anacceleration tracking mirror) is used to compensate for local stageaccelerations that could result in unacceptable pixel smear, thusstabilizing the image. In single tracking mirror embodiments, a singleset of scanning optics is used both to track the movement of a stage atan anticipated velocity or velocity pattern and to compensate for localstage accelerations (i.e. velocity fluctuations) that could result inunacceptable pixel smear, thus stabilizing the image. For singletracking mirror embodiments, a single set of scanning optics compensatesfor all stage motion including velocity and acceleration (or velocityfluctuations). In some embodiments, the single set of scanning opticsincludes a motion tracking mirror to indicate its compensation for bothconstant or anticipated velocity or velocity patterns and measured orpredetermined velocity fluctuations (accelerations).

In some embodiments, the movement of a tracking mirror in response tovelocity fluctuations of the moveable stage is based on a feedbackcontrol mechanism. In some embodiments, the feedback control mechanismcomprises a device to measure position of a substrate over time, such asan encoder. In some embodiments, the movement of a mirror in response tovelocity fluctuations is based on predetermined velocity fluctuationsfor a moveable stage. In some embodiments, all rotatable scanning opticsare positioned along an optical path before any splitter used to splitan image to multiple cameras.

In some embodiments, provided herein are optical scanning devicescomprising a velocity tracking mirror configured to rotate to allow acamera sensor to image a field of a substrate moving along an axis on amoveable stage. The velocity tracking mirror is operably mounted to thedevice to reflect light along an optical path from the objective lens tothe camera.

In order to maintain a still image of a moving substrate, the velocitytracking mirror is configured and operative to move in coordination withthe moveable stage, while the moveable stage moves the substrate in thesame specified direction, in order to reflect light from the objectivelens to the camera. Thus, the velocity tracking mirror can be operablymounted to the device to rotate about a fixed axis. In some embodiments,the fixed axis is parallel to the plane of the 2-dimensional substrateimage. In some embodiments, the fixed axis is orthogonal to the opticalpath. Thus, the velocity tracking mirror is configured and operative toperform an angular motion that allows the camera to acquire a stillimage of a field of the substrate through an objective lens while thesubstrate is being moved by the moveable stage.

The velocity tracking mirror can be operably coupled to an electricalmotor to effect rotation of the velocity tracking mirror. In preferredembodiments, the electrical motor operably coupled to the velocitytracking mirror is a galvanometer, although other types of electricalmotors may be used. An example of a suitable galvanometer is a NutfieldQS-7 OPD Galvanometer Scanner (Nutfield Technology). In someembodiments, other mechanisms to actuate the velocity tracking mirror,such as those based on hydraulics, pneumatics, or magnetic principles,may also be used. In some embodiments, the electrical motor isoperatively coupled to the velocity tracking mirror and is operative toangularly move the velocity tracking mirror in coordination with themoveable stage, while the moveable stage moves the substrate, in orderto keep an image of the substrate (or a field) still with respect to thecamera while the image is being acquired through the objective lens.

The movement of the velocity tracking mirror can be coordinated througha controller module configured to send a driving signal to theelectrical motor operably connected to the velocity tracking mirror. Thecontroller module can include a motion controller component to generatea desired output or motion profile and a drive or amplifier component totransform the control signal from the motion controller into anelectrical signal or a drive signal that actuates the electrical motor.

In some embodiments, the velocity tracking mirror has an angular rangeof rotation of about 60 degrees, 50 degrees, 40 degrees, 30 degrees, 20degrees, 15 degrees, 10 degrees or 5 degrees. In some preferredembodiments, the velocity tracking mirror has an angular range ofrotation of about 3 degrees, 2 degrees, 1 degree, ½ degree, ¼ degree, or1/10 degree.

In an optical scanning system that uses a velocity tracking mirror toimage a moving substrate, the mirror angle is adjusted with time so thata camera can view a fixed area on a moving substrate. This is referredto as the “forward scan” time. The velocity tracking mirror can thenquickly rotate to return to its initial position. This is referred to asa “fly-back” time or “backscan” time. During the fly-back time, theimage projected onto the camera is not stable.

FIG. 2 illustrates a diagram of velocity tracking mirror angularmovement and timing according to an example embodiment. In operation,the objective lens is focused on a substrate (e.g., an array chip) thatis moving along an axis during imaging. FIG. 2 shows movement of thestage over time 210. During this movement, the velocity tracking mirrorrotates from its initial position to its end position to track themovement of the substrate, which is represented as the forward scan time220. During a single forward scan, a portion of the substrate is imaged,which is referred to herein as a field. The rotation of the velocitytracking mirror allows imaging of the substrate portion corresponding tothe field by the camera during the exposure time, thereby allowingsufficient exposure onto the camera sensor. Any remaining movement ofthe field with respect to the camera can be due to velocityfluctuations, or deviations of the substrate velocity from theanticipated velocity. When the velocity tracking mirror reaches itsextreme end position, it then moves back to its initial position inpreparation for a new scan, which is represented by the waveform ormotion of the mirror at 230 (fly-back time). Still images of thesubstrate are not acquired during the fly-back time intervals. Theforward scan and fly-back motions of the velocity tracking mirror arerepresented as a sawtooth waveform (FIG. 2), which reflects both themotion of the velocity tracking mirror during scanning and flyback andthe driving signal sent to an electrical motor operably connected to thevelocity tracking mirror to actuate the mirror.

An embodiment of a sawtooth waveform to drive the mirror (including aforward scan and backscan segments) is shown in FIGS. 2 and 3. In someembodiments, the velocity tracking mirror may have a non-linear responseover segments of it's range of motion. In this case, the velocitytracking mirror response may be linearized by adjusting the waveform ordriving signal so that you to linearize the response from the velocitytracking mirror.

In some embodiments, the velocity tracking mirror is operably coupled toan electrical motor to effect rotation of the acceleration trackingmirror. In preferred embodiments, the electrical motor operably coupledto the velocity tracking mirror is a galvanometer, or electric coil in amagnetic field that moves in response to an electrical current. In someembodiments, other mechanisms to provide actuation of the velocitytracking mirror, such as those based on hydraulics, pneumatics, ormagnetic principles, may also be used. In some embodiments, theelectrical motor operatively coupled to the velocity tracking mirror isoperative to generate angular motion of the velocity tracking mirror asa function of a velocity of the moveable stage or substrate.

In some embodiments, the movement of the velocity tracking mirror iscoordinated through a controller module configured to send a drivingsignal to the electrical motor operably coupled with the velocitytracking mirror. The controller module can include a motion controllercomponent to generate a desired output or motion profile and a drive oramplifier component to transform the control signal from the motioncontroller into energy that is presented to the electrical motor as anelectrical signal or a drive signal.

In some embodiments, the driving signal or electrical signal sent to theelectrical motor operably coupled with the velocity tracking mirror canbe a linearized velocity tracking error waveform defined as a functionof G(O,w,c(0)), where G is a modified triangle wave with O=angularposition, w=frequency, and 6(0)=amplitude.

The movement of a tracking mirror can be characterized by its dutycycle, defined as the portion of time the tracking mirror is operablymoving in the forward scan motion to allow active imaging of thesubstrate. For example, if the tracking mirror tracks the substrate toallow imaging by the camera during at least 90% of the tracking mirrorcycle (e.g., when the tracking mirror fly-back time is equal to or lessthan 10% of the cycle), then this technique allows the camera to operatewith at least a—90% overall readout efficiency.

In some embodiments, such as fluorescence imaging where longer exposuretimes may be needed, the scan time interval, during which an image iscollected by the camera, must be long enough to build up adequatesignal-to-noise ratios as fluorescence imaging light levels aretypically very weak.

The duty cycle is also impacted by the speed with which the trackingmirror returns to its initial position. This fly-back time interval canbe configured to be only a small fraction of the tracking mirror cycle,thus maximizing the duty cycle. For better efficiency, the amount oftime spent by a tracking mirror on each imaged area is made commensuratewith the camera's frame rate, thereby allowing sufficient time to exposean image of each field onto the camera.

In some embodiments, the duty cycle is greater than 60%. In someembodiments, the duty cycle is from 60% to 90%. In some embodiments, theduty cycle of the image capture is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9% or 10%. In some embodiments, the duty cycle can be as low as 10%,or can be in the range of 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. Insome embodiments, these duty cycles are achieved with an imagingfrequency of from 30 to 200 Hz. In some embodiments, these duty cyclesare achieved with an imaging frequency of from 30 to 40 Hz. In someembodiments, these duty cycles are achieved with an imaging frequency of30 Hz, 35 Hz, 40 Hz, 45 Hz, or 50 Hz.

During the fly-back time intervals, the optical scanning system shouldcease imaging because the image being acquired is not stable. Thus, invarious embodiments various mechanisms can be used to prevent imageexposure to the camera during the fly-back time intervals. For example,in some embodiments an acousto-optic modulator (AOM) switch (or othertype of fast switch) may be used to turn on and off the illuminationlight that is incident onto the substrate being imaged. In otherembodiments, a suitable aperture can be placed in the optical path ofthe illumination light, where the illumination light is allowed to overscan, but the aperture prevents the light from illuminating thesubstrate during the fly-back time intervals by blocking out the lightoutside of the field of view. In yet other embodiments, a suitableshutter can be placed in the optical path of the illumination light,where the shutter is kept open during exposure intervals and is closedduring the tracking mirror fly-back time intervals.

In dual tracking mirror embodiments, the optical scanning device furthercomprises an acceleration tracking mirror configured and operative toprovide offset corrections to an optical path to stabilize thetransmission of light from a substrate to the camera during imaging ofthe substrate (or a portion thereof). The offset corrections are afunction of velocity fluctuations in the movement of the moveable stagealong an axis as compared to the velocity tracked by the velocitytracking mirror. These velocity fluctuations can impact the accuracy oftracking of the moveable stage by the velocity tracking mirror andresult in an image with unacceptable pixel smear. The rotation of theacceleration tracking mirror, as provided herein, stabilizes the imageof the field captured by the camera to reduce pixel smear from velocityfluctuations of the stage or substrate.

FIG. 3 provides one example of an acceleration tracking mirror waveformgenerated in response to an average stage velocity error for a field.When the stage velocity has a positive error, an acceleration trackingmirror waveform is generated to track the additional velocity of thestage. Conversely, when the stage velocity has a negative velocityerror, an acceleration tracking waveform is generated to track theslower velocity of the stage (i.e., it rotates in the opposite directionas a positive velocity error). In some embodiments, the accelerationtracking mirror waveform is generated and converted to a driving signalimmediately after sensing the velocity error. In some embodiments, theacceleration tracking mirror waveform is generated based on an averagemeasurement of velocity during imaging of a field n−1, and the drivingsignal is generated from this waveform to drive movement of theacceleration tracking mirror during imaging of field n.

Stage velocity error can be modeled as a function of amplitude (A),stage position (x), and time (t), to give the following function:

-   -   F(A,x,t)=A(x)*Err(x,t)

In some embodiments, the electrical signal or driving signal (D) tocontrol movement of an electrical motor operably connected to theacceleration tracking mirror can be determined based on the stagevelocity error by a function represented as follows:

-   -   D(F,C,x,E)=F(A,x,t)y*C*x+E,

where C is a scaling factor, x=stage position and E is an offset.F(A,x,t)y is the average value of F(A,x,t) over the ramp range=y, orover a prior field, as described herein. A function to smoothdiscontinuities can also be used to generate the acceleration trackingmirror driving signal.

In some embodiments, the acceleration tracking mirror is operablycoupled to an electrical motor to effect rotation of the accelerationtracking mirror. In preferred embodiments, the electrical motor operablycoupled to the acceleration tracking mirror is a piezoelectric actuator,although other types of electrical motors may be used. In someembodiments, other mechanisms to provide actuation of the accelerationtracking mirror, such as those based on hydraulics, pneumatics, ormagnetic principles, may also be used. In some embodiments, theelectrical motor operatively coupled to the acceleration tracking mirroris operative to generate angular motion of the acceleration trackingmirror as a function of fluctuations in the velocity of the moveablestage to compensate for velocity fluctuations during imaging.

In some embodiments, the movement of the acceleration tracking mirror iscoordinated through a controller module configured to send a drivingsignal to an electrical motor operably connected to the accelerationtracking mirror. The controller module can include a motion controllercomponent to generate a desired output or motion profile and a drive oramplifier component to transform the control signal from the motioncontroller into energy that is presented to the electrical motor as anelectrical signal or a drive signal. Since the movement of theacceleration tracking mirror is a function of fluctuations in velocityof the moveable stage, the controller module can further comprise aposition, velocity or acceleration sensor. This sensor can act as a typeof feedback sensor that determines information about the position and/ormotion of the substrate or moveable stage. In some embodiments, thesensor comprises an encoder (e.g., a linear encoder) or aninterferometer operably mounted to the scanning device. In someembodiments, the encoder is a non-interferometric encoder. In someembodiments, an accelerometer could be used to determine changes invelocity. In some embodiments, the sensor is a component that providesinformation from a velocity fluctuation table that includes anticipatedvelocity fluctuation values for a stage to incorporate into the drivingsignal for the electrical motor operably coupled to the accelerationtracking mirror.

An encoder can be a sensor, transducer or readhead paired with a scalethat encodes position. In some embodiments, the sensor reads the scale(e.g., encoder counts) in order to convert the encoded position into ananalog or digital signal, which can then be decoded into position by adigital readout (DRO) or motion controller. Thus, in some embodiments,the position sensor (including position, velocity, and/or accelerationsensors) is a linear encoder that interfaces with encoder counts (oranother scale) on the substrate or moveable stage. In some embodiments,the encoder counts on the substrate are positioned at a distance of 10p.m., 5 μm, 2 p.m., 1 μm, or 500 nm or less between each encoder count.In some embodiments, the resolution of position detectable by theencoder is 1 nm or less. This can be done for example, usinginterpolation between lines on a substrate or between encoder counts.The spacing between encoder counts can correlate with stage scan speedand frequency of position measurement.

In some embodiments, the scale used by an encoder, such as a linearencoder, can be optical, magnetic, capacitive, inductive, based on eddycurrent. In some embodiments, position detection can be done without ascale on the substrate or moveable stage, for example, by using anoptical image sensor based on an image correlation method.

The position measurements from a substrate or stage position or motionsensor are used to provide a set of data that represents the measuredvelocity of the substrate or moveable stage. The measured velocity canbe compared with an anticipated velocity to determine velocityfluctuations in the stage. These velocity fluctuations can then betranslated into an electrical signal (e.g., a driving signal) whicheffects controlled movement of an electrical motor operably connected tothe acceleration tracking mirror. The controlled movement of theacceleration tracking mirror adjusts the position of the optical pathbetween the substrate and the camera to provide an image with increasedstability, increased sharpness, and/or reduced blur or pixel smear.

An electrical motor can be selected on the basis of its ability toquickly respond to a driving signal comprising a correction term basedon measured velocity fluctuations. To provide a quick response, in someembodiments, the electrical motor has a total angular range of rotationof less than one degree. In some embodiments, the electrical motor is apiezoelectric actuator or another motor with a similar response time tothe correction signal. In some embodiments, the position sensor acquiresposition information at a rate of equal to or greater than 500 Hz, 1kHz, 2 kHz, 3 kHz, 4 kHz, 5 kHz, 10 kHz, 20 kHz, 50 kHz, 100 kHz and 250kHz. In certain embodiments, a higher frequency of position detection,e.g., 5 kHz or more, allows a more precise measurement of the stage toincrease the resolution of velocity fluctuation and therefore provide asharper image. However, lower frequencies may be used that aresufficient to provide correction to prevent a pixel smear of greaterthan two pixels.

For example, in some embodiments an encoder provides substrate or stageposition or motion measurement information to logic executing in acomputing device, such as a motion controller, where the logic uses themeasurement information to compute the necessary correction term for thedirection of stage movement and to cause a servo mechanism, such as anelectrical motor, to rotate the acceleration tracking mirror based onthe computed correction term that is a function of velocity fluctuationof the moveable stage.

The determination of velocity fluctuation can be determined from two ormore position measurements from the position sensor. In someembodiments, near instantaneous velocity can determined from the mostrecent 2 or 3 positions measured from the substrate.

In some embodiments, velocity fluctuation used to generate a drivingsignal is determined from a pre-calculated table. A velocity fluctuationmay already be known for a stage, and can be recorded into a table whichis accessed by the motion controller component. Thus, in theseembodiments, the position sensor is a component of the controller modulethat provides data from a velocity fluctuation table to a motioncontroller.

By using an acceleration tracking mirror as described herein, an opticalscanning system can use a camera that operates in a full-frame mode(e.g., such as a CMOS camera that does not operate in TDI mode) toacquire still images of a moving substrate within an accuracy of +/−onepixel. In some embodiments that are employed for biological imaging,e.g., DNA sequencing or other single molecule detection techniques, theextreme alignment accuracy requirements of fluorescence imaging maynecessitate the use of at least one velocity and acceleration trackingmirror pair to correct for movement, including velocity fluctuations, ofa substrate along an axis to remove nonlinearities in the motion of themoveable stage.

In some embodiments, tracking of the movement of the stage, includingboth a velocity of a stage and velocity fluctuations of the stage alongan axis, is performed by a single tracking mirror (the single-mirrorembodiment), referred to herein as a motion tracking mirror. In thisembodiment, a single motion tracking mirror performs the functions ofboth the velocity and acceleration tracking mirrors described above.Therefore, in a single mirror embodiment, a drive signal is sent to anelectrical motor operably coupled to the single motion tracking mirrorthat is a function of a predetermined stage velocity including bothscanning and flyback waveforms (e.g., a sawtooth wave) and is also afunction of velocity fluctuations of the stage or substrate, which canbe predetermined or can be based on one or more measurements thatprovide information about the motion of the substrate or moveable stageto determine a velocity fluctuation of the stage or substrate.

Scanning optics provided in a single mirror embodiment of the opticalscanning system is shown in FIG. 4. In this embodiment, the opticalscanning system comprises a moveable stage 110 configured to move amounted substrate 120 along an axis. The substrate 120 comprises one ormore fields 121 that are individually imaged by the optical scanningsystem as the stage is continuously moving. The substrate is illuminatedby an illumination mechanism (not shown), and light from the substratetravels along an optical path through the objective lens 130. The imageof the moving substrate is stabilized with respect to an image sensor bya motion tracking mirror 145. An image of the field 121 is captured by acamera 160 comprising an image sensor. The motion tracking mirror 145 isconfigured to rotate about an axis parallel to the plane of the imagefield. The rotation of the motion tracking mirror 145 adjusts theoptical path to stabilize the image of a field during an image captureby the camera 160. Rotation of the motion tracking mirror 145 is afunction of both a predetermined stage velocity and velocityfluctuations of the stage or substrate. Thus, the single-mirrorembodiment of the optical scanning system provides a stabilized imagewith an improved sharpness or reduced pixel smear over a system thatdoes not correct for stage velocity fluctuations while imaging a movingsubstrate.

A controller module configured to drive the movement of the singlemotion tracking mirror includes components of both a controller moduleoperably connected to a velocity tracking mirror and components of acontroller module operably connected to an acceleration tracking mirror,as described in the two-mirror embodiment above. Therefore, in someembodiments, the controller module comprises a motion controllercomponent that generates a desired output or motion profile, a drive oramplifier component to transform the control signal from the motioncontroller into an electrical signal or drive signal. The controllermodule can also include a position, velocity, or acceleration sensorconfigured to determine the position or motion of the substrate ormoveable stage, and to send this signal to the motion controllercomponent to be used to generate the desired output or motion profile asa function of the information from the sensor. The motion controllercomponent can then generate a motion profile for the single motiontracking mirror that is a function of both the constant or otherwiseanticipated velocity of the substrate or stage (e.g., a sawtoothwaveform) and velocity fluctuations determined from the signal from thesensor or that are predetermined for the stage. Thus, the sawtoothwaveform used to track velocity can be modified according to a real timevelocity measurement determined from a signal from a positional sensoror from predetermined velocity fluctuations.

FIG. 5 provides an example stage velocity error waveform generated fromdata provided by a position, velocity or acceleration sensor. Also shownare approximate velocity corrections needed, otherwise known as theaverage velocity error for a field. The next waveform (solid line) showsa velocity tracking waveform modified by a correction term that is afunction of an average velocity error (e.g., an average velocity errorfor a field). The dashed line represents a linearized, un-correctedmotion tracking mirror waveform (similar to the waveform used to drive avelocity tracking mirror in the dual-mirror embodiment). When there is apositive velocity error, the slope of the waveform during scanning isincreased, thereby increasing the speed of the rotation of the mirror tocompensate for the velocity error. When there is a negative velocityerror, the slope of the waveform is decreased, thereby decreasing thespeed of the rotation of the mirror to compensate for the velocityerror.

In some embodiments, the sensor determines an average velocity of thestage or substrate based on a plurality of measurements taken duringimaging of a field. In some embodiments, the sensor is a positionsensor. In some embodiments, the position sensor is an encoder (e.g., alinear encoder) or an interferometer operably mounted to the scanningdevice. The signal from the position sensor can be used to determine theaverage velocity of the moveable stage or substrate using two or more ofthe most recent positional measurements captured by the position sensor.In some embodiments, these measurements can be used to adjust the angleof the motion tracking mirror after sensing.

In some embodiments, the average velocity of a substrate or stage isdetermined over a field n−1 and is used to provide a correction termwhich is used for generating the motion profile of the motion trackingmirror for field n. This is known as the field level feed forwardmechanism, as illustrated in FIG. 6. In some embodiments of the fieldlevel feed forward correction mechanism, the motion controller componentgenerates a motion profile for movement of the motion tracking mirror(in the single mirror embodiment) or the acceleration tracking mirror(in the dual mirror embodiment) as a function of the average velocity ofthe previous imaged field. A field level feed-forward velocity trackingand correction mechanism is distinct from other types of correction,such as a scanline level feed forward mechanisms. Field-level feedforward corrections are advantageous in that they reduce the stringencyof immediate signal processing while still providing sufficientcorrection information to generate an acceptably sharp image formonitoring information in single pixels (i.e., a pixel smear of no morethan +/−one pixel). Some image blur or pixel smear may not be correctedby field level feed forward mechanisms, however, in some embodiments,such as in single molecule imaging applications (e.g., for biomolecularsensing), a pixel smear of up to +/−one pixel is acceptable, andfield-level feed forward corrections can generate an acceptable smearwhen there is a velocity fluctuation from field n−1 to field n that iswithin an acceptable level (e.g., results in a pixel smear of no morethan +/−one pixel).

Provided in FIG. 6 is a diagram of an embodiment of field levelfeed-forward correction. In this embodiment, velocity trackingmeasurements of a chip or stage are obtained to generate a mirrorrotation drive signal that incorporates velocity fluctuations of themoving stage. Here, the stage begins moving, and positional informationis obtained over time (or velocity information is obtained) to determinea pre-field non-linearity of the velocity of the stage (velocityfluctuations). When the first field is imaged, a driving signal that isa function of the average velocity measured in the pre-field stage issent to the motion tracking mirror (or acceleration tracking mirror intwo-mirror embodiments). For the next consecutive fields, the process isrepeated using velocity error determined from positional information ofthe prior field (N−1) over time. The driving signal is determined as afunction of this velocity error and sent to the motion tracking mirrorfor rotation during field N. FIG. 6 shows stage velocity error over timeand also the approximate velocity error per field. The first arrow downfrom the stage velocity error indicates that determination of an averagevelocity error from the field, and the translation into a “same field”acceleration correction waveform. The feed forward mechanism isindicated by the second arrow down, translating this waveform to drivethe mirror for the next field n based on the waveform derived from fieldn−1. In this manner, the stage velocity error is approximated for eachfield based on the prior field n−1.

In one embodiment, the field level feed forward mechanism proceedsaccording to the following steps:

-   -   a) Measure multiple positions of the substrate over field n−1.    -   b) Determine an average velocity for field n−1.    -   c) Calculate the velocity fluctuation for field n−1 and a        correction term based on this velocity fluctuation.    -   d) Apply correction term to motion profile (e.g., an electric        motor waveform) to send to driver or amplifier.    -   e) Send a driving signal to an electric motor operably linked to        a motion tracking mirror or acceleration tracking mirror to        generate movement of the tracking mirror during image capture of        field n.    -   f) Repeat process for remaining fields in lane

In some embodiments, the total feedback loop in the servomechanism basedon field level feed-forward velocity tracking is less than 100 ms, lessthan 90 ms, less than 80 ms, less than 70 ms, less than 60 ms, less than50 ms, less than 40 ms, less than 30 ms, less than 20 ms, less than 10ms, less than 5 ms, or less than 2 ms. In some embodiments, feed-forwardvelocity tracking is used to adjust the movement of an accelerationtracking mirror in the two mirror optical path alignment correctionembodiment.

In some embodiments, in order to minimize error due to the linear rampof an electric motor-controlled single mirror, the electric motordriving signal or waveform is adjusted to compensate for systematicerrors in tracking. Minimization of error in generating a linear ramp(e.g., a forward scan or fly-back) of an electric motor can also beachieved by reducing the speed of motion of the mirror, such as byreducing the imaging frequency of the optical scanning system. In someembodiments, the frequency of the sawtooth waveform to control theelectric motor in the single mirror embodiment is kept at or below 200Hz. In some embodiments, the frequency of the sawtooth waveform tocontrol the electric motor in the single mirror embodiment is from 50 Hzto 30 Hz. In some embodiments, the frequency of the sawtooth waveform tocontrol the electric motor in the single mirror embodiment is from 45 Hzto 35 Hz. In some embodiments, the duty cycle of the sawtooth waveformto control the electric motor in the single mirror embodiment is 70% orless. In some embodiments, the duty cycle of the sawtooth waveform tocontrol the electric motor in the single mirror embodiment is from 60%to 80%. In some embodiments, the frequency of image capture and the dutycycle in the single mirror embodiment are adjusted to have a totalvelocity tracking error of less than 2%. In some embodiments, thefrequency of image capture and the duty cycle in the single mirrorembodiment are adjusted to have a total pixel smear of less than 2pixels or less than 1 pixel.

As discussed herein, according to some embodiments, the controllermodule refers to a collection of components including i) sensors todetermine states of parts of the optical scanning system (e.g., a stageposition sensor) for feedback control, ii) mechanisms that calculate orotherwise provides waveforms for effecting movement of components of theoptical scanning device (e.g., a sawtooth wave to drive a velocitytracking mirror), or iii) mechanisms that send a driving signal to anactuator based on the waveform to effect movement of a component.

For example, as discussed above, the controller module can be used tocreate the correct waveforms to drive the movement of certaincomponents, such as rotatable mirrors to adjust the optical path, andsynchronize them to stage motion based on stage encoder or master clockvalues. The waveform for a velocity tracking mirror can be a sawtoothwaveform with a ramp that tilts the velocity tracking mirror at theright speed to match of the velocity of the stage. The waveform sent toan acceleration tracking mirror or to a single rotatable mirror in thesingle mirror embodiment must include a term to correct for velocityfluctuations that occur in the moveable stage velocity. This waveformcan be created by “mapping” out the stage velocity non-linearities usinga reticle with calibration marks on it, or it can be created by takingthe measured stage velocity from the previous field, creating a waveformthat compensates for velocity non-linearities and using that waveform tocorrect for velocity fluctuations in the next field, i.e., the fieldlevel “feed-forward” approach. The waveform can also be created byproviding information from a velocity fluctuation table to thecontroller module.

According to the techniques described herein, one or more computingdevices and/or various logic thereof are configured and operative tocontrol the coordinated motions of the scanning mirror or mirrors (e.g.,the acceleration and velocity tracking mirrors) and the moveable stage.Thus, in some embodiments the moveable stage (and therefore thesubstrate mounted thereon) can be configured to move with constantvelocity, in which case the back-scan motion of the tracking mirror willalso be at a suitable constant velocity. In other embodiments, themoveable stage can be configured to move with non-constant velocity, inwhich case the back-scan motion of the tracking mirror will also be at asuitable non-constant constant velocity.

The controller module can also be used to synchronize components of theoptical scanning device to enable capture of an image of a field of asubstrate on a moving stage. In addition to linking motion of therotatable mirrors to the velocity of a moveable stage, the controllermodule can also control other components of the device. In someembodiments, the controller module comprises a mechanism to controlillumination of the field. For example, the controller module may send asignal to an illumination device, such as a laser, to time illuminationwith the image capture process. In some embodiments, illumination stateis dependent upon the sawtooth waveform sent to a velocity trackingmirror. In some embodiments, the controller module sends a signal tocontrol movement of the moveable stage at a selected velocity or along aselected path, such as a serpentine path to image several fields on asubstrate.

The connection of the controller module to certain components of anoptical scanning system, according to a dual-mirror embodiment, is shownin FIG. 7A. As illustrated in this embodiment, the controller module isoperably connected to an illumination component to control illuminationof a substrate, such as by timing illumination with image capturetiming. The controller module is operably connected to a camera tocontrol image capture by the camera to coordinate with the motion of therotating mirrors, e.g., such that an image is acquired during trackingof each field, and no image is acquired during the fly-back period of atracking mirror. As described in more detail herein, the controllermodule can comprise a memory, a processor, and a driver. The memory canhold a predetermined velocity or velocity fluctuation information to beused by the processor to generate a waveform. The memory can also hold apredetermined waveform. The waveform can be sent to the driver togenerate a driving signal. In some embodiments, the controller module isoperably connected to an encoder (e.g., a linear encoder) to receivepositional information about the moving stage over time. The controllermodule can then generate a drive signal as a function of velocityfluctuation from the information from the linear encoder, which can thenbe sent to the driver to send a driving signal to an accelerationtracking mirror (or substrate tracking mirror in the one-mirrorembodiment (FIG. 7B)). The path from data collection from the stage tomovement of a tracking mirror is indicated by the dotted arrows, whichalso include a driving signal sent from the controller module to a motoroperably connected to an acceleration tracking mirror 150 or motiontracking mirror 145. FIGS. 7A and 7B also depicts the optical path oflight from an illumination source to detection by the camera accordingto a dual-mirror embodiment. Solid lines (not arrows) in FIGS. 7A and 7Bindicate an operable connection between a motor and a component of thedevice actuated by the motor.

In an example embodiment, the optical scanning system further comprisesan illumination light source. In various embodiments, the illuminationsource can emit light of various wavelengths that are compatible withvarious fluorophores that can be used in biomolecular detection, forexample, light of wavelength in a range from 400 nm to 800 nm. In someembodiments, the illumination source is mounted underneath thesubstrate, such that light collected by the objective lens istransmitted through the field to the objective lens. In otherembodiments, the illumination source is mounted above the substrate,such that light collected by the objective lens is reflected by thefield to the objective lens.

The optical scanning system can further comprise a dichroic mirror. Inan example embodiment, the optical scanning system further comprises anillumination source and a dichroic mirror, where the dichroic mirror isconfigured and operative at least to: (a) reflect light from theillumination source to illuminate a field of the substrate or a portionthereof; and (b) pass through light that is emitted by the sample andpasses through the objective lens.

In some embodiments, the optical scanning system further comprises asplitter. The splitter can be placed along the optical path after theacceleration and velocity tracking mirrors (or single tracking mirror)to split the optical signal comprising the field image to two or morecameras.

The optical scanning system can also comprise a tube lens componentpositioned in an optical path between the tracking mirror and theobjective lens, so that the tracking mirror can be situated at the pupilof the objective lens. Relay lenses or tube lenses may also be usedalong the optical path at other locations to invert an image or toextend the optical path.

In some embodiments, the optical scanning system comprises a relay lenssystem used to create a region in the optical path which has all raysnominally parallel and also has a small beam diameter. In someembodiments, scanning optical elements are placed where the optical pathhas a small beam diameter to ensure that their placement: (i) minimizespower loss, (ii) minimizes image degradation and (iii) minimizes thesize of the optical elements so that their mass can be as small aspossible. This enables higher scanning frequencies and a lighter weightsystem.

The use of a relay lens system can facilitate fluorescence-based opticalscanning systems that are used for biomolecular detection on asubstrate, as these systems typically employ very low light levels withdim fluorescence images. Thus, relay lenses are effective to increasethe efficiency and sensitivity of the optical scanning system to keepimage acquisition time to a minimum. Further, in some embodiments,illumination intensity must remain below the point where it can damagebiomolecules on the substrate.

FIG. 8A illustrates an example method for imaging a substrate accordingto a dual-mirror embodiment. The method in FIG. 8A is not limited tobeing performed by any particular type of machine or device, andtherefore the method description hereinafter is to be regarded in anillustrative rather than a restrictive sense.

In step 810, a moveable stage moves a substrate under an objective lensin a plane that is normal to the optical axis of the objective lens.While the substrate is in motion, in step 820, a servo mechanism (e.g.,an electric motor) changes the angle of a velocity tracking mirror totrack the velocity of the moving stage during the capture of an image ofa field of the substrate. In some aspects, a controller module that ispart of or coupled to, the velocity tracking mirror executes logic thatcontrols the servo mechanism operably connected to the velocity trackingmirror. In step 840 a servo mechanism changes the angle of anacceleration tracking mirror to track velocity fluctuations of themoving stage during the capture of an image of a field of the substrate.In some aspects, a controller module that is part of or coupled to, theacceleration tracking mirror executes logic that controls the servomechanism in coordination with the moveable stage. In some embodiments,logic receives feedback control information that represents the movement(e.g., velocity fluctuations) of the moveable stage and uses thisinformation to adjust the input signal to the servo mechanism, which inturn changes the angle of the acceleration tracking mirror, therebysynchronizing the combined motion of the velocity tracking mirror andacceleration tracking mirror with the movement of the moveable stage. Insome aspects, this feedback information is received 831 from an linearcontroller that detects whether there are any nonlinearities in themotion of the moveable stage 830. The logic then uses this informationto compute offset corrections and passes the offset corrections as aninput signal to a servo mechanism that controls the angle of theacceleration tracking mirror in the optical path between the trackingmirror and the camera. In this manner, by making minor adjustments tothe angle of the acceleration tracking mirror, the logic effectivelyremoves from the image being acquired any errors that are caused bynonlinearities in the motion of the moveable stage.

In step 850, the camera records the still image of the substrate (or aportion thereof) while the substrate is being moved by the moveablestage.

FIG. 8B illustrates an example method for imaging a substrate accordingto a single-mirror embodiment. The method in FIG. 8B is not limited tobeing performed by any particular type of machine or device, andtherefore the method description hereinafter is to be regarded in anillustrative rather than a restrictive sense.

In step 810, a moveable stage moves a substrate under an objective lensin a plane that is normal to the optical axis of the objective lens,where the substrate comprises a multitude of distinct features that arethe targets of the imaging.

While the substrate is in motion, in step 845 a servo mechanism changesthe angle of a motion tracking mirror to track velocity fluctuations ofthe moving stage during the capture of an image of a field of thesubstrate. In some aspects, a controller module that is part of orcoupled to, the motion tracking mirror executes logic that controls theservo mechanism in coordination with the moveable stage. In someembodiments, logic receives feedback control information that representsthe movement (e.g., velocity fluctuations) of the moveable stage anduses this information to adjust the input signal to the servo mechanism,which in turn changes the angle of the motion tracking mirror tocompensate for velocity fluctuations of the moveable stage. In someembodiments, the controller module incorporates the velocity fluctuationof the moveable stage into a sawtooth waveform for tracking apredetermined velocity, which is used as a driving signal to controlmovement of the motion tracking mirror. In some aspect, this feedbackinformation is received 831 from an linear controller that detectswhether there are any nonlinearities in the motion of the moveable stage830. The logic then uses this information to compute offset correctionsand passes the offset corrections as an input signal to a servomechanism that controls the angle of the motion tracking mirror in theoptical path. In this manner, by making minor adjustments to the angleof the motion tracking mirror, the logic effectively removes from theimage being acquired any errors that are caused by nonlinearities in themotion of the moveable stage.

In step 850, the camera records the still image of the substrate (or aportion thereof) while the substrate is being moved by the moveablestage.

Optical scanning systems provided herein compensate for stage velocity(or any other imaging of moving components) non-linearities (e.g., localstage accelerations) that would normally result in a blurry image in adevice that tracks only stage velocity, but does not have a mechanism tocompensate for stage velocity fluctuations. In some embodiments, theoptical scanning system is capable of generating stabilized images of acontinuously moving substrate or other object at 30 frames per second.In some embodiments, the optical scanning system is capable ofgenerating still images of a continuously moving substrate or otherobject at from 10 to 30 frames per second. In some embodiments, theoptical scanning system is capable of generating still images of acontinuously moving substrate or other object at 40 frames per second.In some embodiments, the optical scanning system is capable ofgenerating still images of a continuously moving substrate or otherobject at more than 30 frames per second, 40 frames per second, 50frames per second, 60 frames per second, 70 frames per second, 80 framesper second, 90 frames per second, 100 frames per second, 120 frames persecond, 150 frames per second or 200 frames per second.

In some embodiments, the stage velocity fluctuation of the opticalscanning system is greater than +/−0.5%. In some embodiments, the stagevelocity fluctuation of the optical scanning system is greater than+/−0.1%. In some embodiments, the stage velocity fluctuation of theoptical scanning system is greater than +/−0.1%, and is reduced to lessthan +/−0.1% as observed by the camera.

In some embodiments, the stage velocity fluctuation of the opticalscanning system is greater than +/−1%. In some embodiments, the stagevelocity fluctuation of the optical scanning system is greater than+/−1%, and is reduced to less than +/−1% as observed by the camera.

In some embodiments, the optical scanning system described hereinprovides an increased sharpness of an image over a system that does notcompensate for velocity fluctuations in a continuously moving stage.

In some embodiments, the total distance a substrate moves during theimaging of a field deviates by more than +/−1 pixel (as measured by theimage of the substrate projected onto the sensor) from a predeterminedmovement based on an anticipated velocity during a capture of a fieldimage, while the optical scanning system generates an image with a pixelblur of less than 1. In some embodiments, a pixel is correlated to anarea of the field of—150 nm×150 nm. In some embodiments, a pixel iscorrelated to an area of the field of—162.5 nm×162.5 nm. In someembodiments, a pixel is correlated to an area of the field that isgreater than the size of a single fluorophore.

Pixel smear is one measure of image sharpness and refers to an imageartifact that results from the movement of a substrate in an opticalfield with respect to an image sensor. One way to measure pixel smear isby looking at the ratio of the major and minor axes of a single spot,also known as the eccentricity. In some embodiments, the eccentricity ofan image generated by the optical scanning system is less than 3. Insome embodiments, the eccentricity of the image is reliable singlefluorophore detection.

FIGS. 9A and 9B provides an example of pixel smear and eccentricity of aresulting image of a substrate from the optical scanning system providedherein. The blue spot represents a single illuminated fluorophore, andeach square is a pixel of—162 nm. Shown in FIG. 9A is an example of apixel smear of +1 pixel, within a preferred range of +/−1 pixel, with aneccentricity of 2. Shown in FIG. 9B is an example of a pixel smear of +2pixels, outside of the preferred range of +/−one pixel, with aneccentricity of 3.

FIG. 10 illustrates a system environment for transferring information toor from the optical scanning device. The system environment can includeone or more client devices 1010, one or more servers 1030, a database1005 accessible to the server 1030, where all of these parties areconnected through a network 1020. In other embodiments, different and/oradditional entities can be included in the system environment.

The system environment allows the results from the optical scanningdevice 1040 to be shared via network 1020 with one or more other usersat their client devices 1010. Results can also be uploaded to the web.

The network 1020 facilitates communications between the components ofthe system environment. The network 1020 may be any wired or wirelesslocal area network (LAN) and/or wide area network (WAN), such as anintranet, an extranet, or the Internet. In various embodiments, thenetwork 1020 uses standard communication technologies and/or protocols.Examples of technologies used by the network 1020 include Ethernet,802.11, 3G, 4G, 802.16, or any other suitable communication technology.The network 1020 may use wireless, wired, or a combination of wirelessand wired communication technologies. Examples of networking protocolsused for communicating via the network 1020 include multiprotocol labelswitching (MPLS), transmission control protocol/Internet protocol(TCP/IP), hypertext transport protocol (HTTP), simple mail transferprotocol (SMTP), and file transfer protocol (FTP). Data exchanged overthe network 1020 may be represented using any suitable format, such ashypertext markup language (HTML) or extensible markup language (XML). Insome embodiments, all or some of the communication links of the network1020 may be encrypted using any suitable technique or techniques.

The client device(s) 1010 are computing devices capable of receivinguser input as well as transmitting and/or receiving data via the network1020. In one embodiment, a client device 1010 is a conventional computersystem, such as a desktop or laptop computer. Alternatively, a clientdevice 1010 may be a device having computer functionality, such as apersonal digital assistant (PDA), a mobile telephone, a smartphone oranother suitable device. A client device 1010 is configured tocommunicate via the network 1020.

In some embodiments, the system environment may include one or moreservers, for example where the diagnostic system is includes a servicethat is managed by an entity that communicates via the network 1020 withthe optical scanning device 1040 and/or any of the client devices 1010.The server 1030 can store data in database 1005 and can access storeddata in database 1005. The server 1030 may also store data in the cloud.In some embodiments, the server 1030 may occasionally push updates tothe optical scanning device 1040, or may receive result data from theoptical scanning device 1040 and perform certain analyses on that resultdata and provide the analyzed data back to the optical scanning device1040 or to a client device 1010.

In some embodiments, the optical scanning device 1040 functionality canbe included in a client device 1010, such as a mobile phone, and can beoperated via a mobile application installed on the phone. The mobileapplication stored on the phone can process the results read from theoptical scanning device and share the results with other devices 810 onthe network 820.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the invention described herein. The scopeof the present invention is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the term “consistingof” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anynucleic acid or protein encoded thereby; any method of production; anymethod of use; etc.) can be excluded from any one or more claims, forany reason, whether or not related to the existence of prior art.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

Section and table headings are not intended to be limiting.

1.-67. (canceled)
 68. An optical scanning system for imaging a movingsubstrate, comprising: a. a stage configured to: i. undergo movementalong an axis, and ii. support a substrate comprising a plurality offields during said movement along said axis; b. a camera in opticalcommunication, through an optical path, with a field of said pluralityof fields, when said substrate is supported by said stage, and whereinsaid camera is configured to capture an image of said substratecomprising said field; c. a velocity tracking mirror and an accelerationtracking mirror mounted along said optical path, wherein said velocitytracking mirror and said acceleration tracking mirror are configured tomaintain said optical path to stabilize said image of said substrateduring said movement of said substrate.
 69. The system of claim 68,further comprising a first electrical motor, and wherein a movement ofsaid velocity tracking mirror is actuated by said first electricalmotor.
 70. The system of claim 69, wherein said first electrical motoris a galvanometer.
 71. The system of claim 68, further comprising asecond electrical motor, wherein a movement of said accelerationtracking mirror is actuated by said second electrical motor.
 72. Thesystem of claim 71, wherein said second electrical motor is apiezoelectric actuator.
 73. The system of claim 68, wherein saidvelocity tracking mirror and said acceleration tracking mirror areadjacent.
 74. The system of claim 68, further comprising an objectivelens, wherein said objective lens is configured to move along saidoptical path and wherein a movement of said objective lens is a functionof a movement of said field out of a focal plane of said objective lens.75. The system of claim 74, further comprising a third electrical motorand wherein a movement of said objective lens is actuated by said thirdelectrical motor.
 76. The system of claim 68, wherein a movement of saidvelocity tracking mirror is a function of a velocity measurement of saidmovement of said stage along said axis.
 77. The system of claim 76,wherein said movement of said acceleration tracking mirror is a functionof a change in velocity measurement of said movement of said stage alongsaid axis.
 78. The system of claim 77, wherein said function of saidvelocity measurement and said function of said change in velocitymeasurement are generated by a linear displacement sensor, wherein saidlinear displacement sensor determines a positional measurement of saidstage.
 79. The system of claim 78, wherein said linear displacementsensor is a linear encoder.
 80. The system of claim 68, wherein saidoptical path comprises a filter configured to reduce a transmission ofexcitation light to said camera.
 81. The system of claim 80, wherein alight source configured to generate said optical path is displacedadjacent to said stage and is not adjacent to said camera.
 82. Thesystem of claim 68, further comprising an additional pair of mirrorscomprising a second velocity tracking mirror and a second accelerationtracking mirror, wherein said additional pair of mirrors is mountedalong said optical path and wherein said additional pair of mirrors isconfigured to motion along an additional axis.
 83. A method of imaging amoving substrate, wherein said substrate comprises one or more fields,the method comprising: a. disposing said substrate on a stage; b.configuring a camera to be in optical communication with said one ormore fields of said substrate by an optical path; c. actuating amovement of said stage and configuring a velocity tracking mirror and anacceleration tracking mirror to maintain said optical path to stabilizean image of said substrate during a movement of said stage; and d.concurrent with said movement, imaging said field passing through anobjective lens using said camera.
 84. The method of claim 83, wherein(d) further comprises actuating a movement of said objective lens alongsaid axis and wherein said movement of said objective lens is a functionof a movement of said one or more fields out of a focal plane of saidobjective lens.
 85. The method of claim 83, wherein (c) comprisesactuating a movement of said velocity tracking mirror.
 86. The method ofclaim 85, wherein said movement of said velocity tracking mirror is afunction of an anticipated velocity of said stage.
 87. The method ofclaim 85, wherein said movement of said velocity tracking mirror is afunction of a velocity measurement of said movement of said stage alongsaid axis.
 88. The method of claim 85, wherein (c) comprises actuating amovement of said acceleration tracking mirror.
 89. The method of claim88, wherein a movement of said acceleration tracking mirror is afunction of a change in velocity measurement of said movement of saidstage along said axis.
 90. The method of claim 89, wherein (c) comprisesconfiguring a linear displacement sensor to generate said function ofsaid velocity measurement and said function of said change in velocitymeasurement of said stage.
 91. The method of claim 83, wherein (b)further comprises displacing a light source configured to generate saidoptical path is displaced adjacent to said stage and is not adjacent tosaid camera.
 92. The method of claim 83, wherein (b) further comprisesdisplacing a filter along said optical path.
 93. The method of claim 83,wherein (c) further comprises actuating a movement of an additional pairof mirrors, said additional pair of mirrors comprising a second velocitytracking mirror and a second acceleration tracking mirror, wherein saidadditional pair of mirrors is mounted along said optical path andwherein said additional pair of mirrors is configured to motion along anadditional axis.